Nitric oxide, a ubiquitous messenger molecule with important regulatory functions, is synthesized by a family of enzymes called nitric oxide synthases (NOS). Three NOS isoforms have been identified: two constitutive, the neuronal (nNOS, type I) and endothelial (eNOS, type III) enzymes, and one inducible (iNOS, type II). All have an amino-terminal heme- and arginine-binding domain, a central calmodulin-binding region, and a carboxyl-terminal reductase domain, with an NADPH-binding site. The eNOS gene, located on chromosome 7q35-36, comprises 26 exons spanning 21 kb. In view of the physiological and pathophysiological importance of NO, the possible role of eNOS in the pathogenesis of various human diseases has been examined using its polymorphic variants as potential disease markers.An eNOS polymorphism in exon 7 (894 G/T) resulting in glutamate or aspartate, respectively, at position 298 of the protein is correlated with severity of cardiopulmonary diseases. Because glutamate and aspartate are considered to be conservative replacements, the polymorphism was thought to be a marker for a functional locus elsewhere in the gene. We now show in transfected cells, primary human endothelial cells, and human hearts, that eNOS with aspartate, but not glutamate, at position 298 is cleaved, producing 100-kDa and 35-kDa products. Recombinant or native eNOS was examined by immunoblotting either in lysates (COS7) or after partial purification over 2',5'-ADP-Sepharose and calmodulin-Sepharose. Immunoblotting after SDS/PAGE with a carboxyl-terminal antibody showed a single major protein band in the predicted position for eNOS at 135 kDa. An additional band of approximately 100 kDa was present only in the recombinant 298Asp eNOS and in the eNOS synthesized by primary cells and heart tissue with a G/T genotype. Using an eNOS amino-terminal-specific antibody, an immunoreactive band at approximately 35 kDa, corresponding to the residual N-terminal cleavage fragment, was observed in those cells with a T genotype. Thus, the eNOS gene with polymorphisms at nucleotide 894 yields protein products with differing susceptibility to cleavage, suggesting that, in contrast to prior predictions, this polymorphism has a functional effect on the eNOS protein.